Inwardly rectifying potassium (Kir) channels are key regulators of diverse physiological processes and may represent novel drug targets for diseases. Their therapeutic potential has not been tested directly, however, due to the lack of drug-like compounds targeting inward rectifiers. The lack of selective probes has also hindered efforts to define the physiological functions of some Kir channels. To overcome this formidable barrier and create new opportunities for studying inward rectifier physiology, the investigators performed a high- throughput screen (HTS) of more than 200,000 compounds for small-molecule modulators of ROMK (Kir1.1), a putative target for a novel class of diuretic. One compound, termed VU590, inhibits ROMK at nanomolar concentrations and Kir7.1 in the low micromolar range, making it the first small-molecule inhibitor of both channels. The investigators went on to use medicinal chemistry to rationally design a nanomolar-affinity probe, termed VU591, which is highly selective for ROMK over more than 60 potential off targets, including inward rectifiers and BK channels. In Aim 1, the investigators will employ state-of-the-art molecular modeling techniques, atomic structure-guided mutagenesis and electrophysiology to define the VU590/591 binding sites in ROMK and Kir7.1. VU591 is remarkably selective for ROMK and therefore represents a promising candidate for further development for use in animal studies. In Aim 2, the investigators will first determine if VU591 is active in the native tissue by assessing its effects on K and Na transport in isolated perfused cortical collecting ducts under low- and high-flow conditions. The investigators also discovered a nanomolar-affinity inhibitor of a G-protein regulated inward rectifier (GIRK), a putative therapeutic target for atrial fibrillation. In Aim 3, the investigators will use medicinal chemistry, structure-guided mutagenesis and electrophysiology to define the molecular binding sites for this novel compound termed VU592. These studies will provide important new insights into the atomic structures of inward rectifiers and generate critically needed probes with which to define the integrative physiology and therapeutic potential of these channels. Lay summary: The investigators will combine medicinal chemistry, advanced computational techniques and classical physiological methods to develop drug-like compounds targeting potassium channels that could be therapeutic targets for hypertension, edema and cardiac arrhythmia.